Safe and efficient gene delivery vectors are essential for the treatment of diseases (Friedmann & Roblin (1972) Science 175:949-955; Mulligan (1993) Science 260:926-932). Vector targeting, efficiency, and safety are of particular importance (Wickham (2003) Nat. Med. 9:135-139; Thomas, et al. (2003) Nat. Rev. Genetics 4:346-358; Ferber (2001) Science 294:1638-1642). Viruses have evolved sophisticated mechanisms to overcome the numerous intracellular barriers encountered in gene delivery including cell entry, nuclear import, and gene expression. Viral vectors, therefore, are highly efficient and are used in the majority of ongoing gene therapy trials (Thomas & Klibanov (2003) Appl. Microbiol. Biotechnol. 62:27-34).
Methods have been suggested for modifying retroviruses to improve retroviral vector infection, replication, and growth.
For example, U.S. Pat. No. 6,846,628 discloses a method for identifying a nucleic acid molecule that interacts with a selection molecule to provide a replication and/or growth advantage greater than that provided a parental molecule.
U.S. Pat. No. 6,713,279 discloses a method of obtaining novel polynucleotides and encoded polypeptides by use of non-stochastic methods of directed evolution for use in evolving genetic vaccines that exhibit increased efficacy.
U.S. Pat. No. 6,596,539 teaches a method for modifying a phenotype of a virus, such as viral tropism and host range, by iterative sequence recombination of variant viruses and selection of improved variants.
U.S. Pat. No. 6,096,548 discloses a method of directing evolution of a virus to increase the efficiency with which it infects a host cell. This reference teaches directed evolution of genes involved in transfer, integration, stability or expression of the vector containing them.
Murine leukemia virus (MLV), the most commonly used retroviral vector in gene therapy clinical trials, has an infectivity half-life (t1/2) in the range of 5 to 8 hours at 37° C. (Kotani, et al. (1994) Hum. Gene Ther. 5:19-28; Andreadis, et al. (1997) J. Virol. 71:7541-7548; Le Doux, et al. (1999) Biotechnol. Bioeng. 63:654-662; Merten (2004) J. Gene Med. 6:S105-S124). This instability limits both virus production and the maximum achievable virus titer as the decay of active virus particles competes with the rate of their generation by producer cells (Merten, et al. (2004) supra; Le Doux, et al. (1999) Biotechnol. Bioeng. 63:654-662). The instability also limits gene transfer efficiency, increasing the dose of virus required to transduce a given number of target cells, thereby increasing costs and raising safety concerns. A more stable retrovirus vector is needed to improve transduction efficiency and virus production, leading to important practical implications. The present invention meets this need in the art.
Should include references teaching the envelope protein as source of instability? I see that this is mentioned in the examples, so maybe not necessary to include here.